Viral interleukin-6

ABSTRACT

The present invention relates to viral interleukin-6 (v-IL-6), which can be obtained by recombinant expression of the DNA of human herpesvirus type 8 (HHV-8), and which may be used in diagnosis and treatment of human diseases such as kaposi sarcoma, Castleman&#39;s disease, multiple myeloma, kidney cell carcinoma, mesangial proliferative glomerulonephritis or B cell lymphoma.

[0001] The present invention relates to viral interleukin-6 (v-IL-6), which can be obtained by recombinant expression of the DNA of human herpesvirus type 8 (HHV-8), and which may be used in diagnosis and treatment of human diseases such as kaposi sarcoma, Castleman's disease, multiple myeloma, kidney cell carcinoma, mesangial proliferative glomerulonephritis or B cell lymphoma.

[0002] Kaposi's sarcoma (KS), a multifocal proliferative lesion of uncertain pathogenesis, is highly prevalent among homosexual AIDS patients. Studies with biopsy materials and cultured cells have indicated an important role of growth factors and cellular cytokines, such as basic fibroblast growth factor, interleukin-1β, platelet derived growth factor, interleukin-6 (IL-6), and oncostatin M for the proliferation of spindle cells in KS^(1,2). Several groups found indication for the expression of interleukin-6 (IL-6) receptors in AIDS-KS cells³ and derived spindle cell lines⁴. As epidemiological evidence had suggested that an infectious agent other than HIV may also be involved in KS pathogenesis, it stirred considerable interest when Chang and colleagues⁵ found DNA sequences of a novel herpesvirus in AIDS-KS tissues. Meanwhile, DNA of this virus was consistently found in all epidemiological forms of KS. The new virus, termed human herpesvirus 8 (HHV-8), shows marked sequence homology to herpesvirus (h.) saimiri, the prototype of γ₂-herpesviruses; thus HHV-8 appears to be the first human member of γ₂-herpesviruses (genus rhadinovirus). Cloning HHV-8 DNA from KS tissues and sequencing indicates a genome organization that is generally collinear to h. saimiri ⁶.

[0003] In the course of these studies we surprisingly found, adjacent to a dihydrofolate reductase gene, an open reading frame (ORF) with the coding capacity for a 204 amino acid polypeptide with marked homology to mammalian IL-6 (P-value for homology searches with NCBI-BLAST: P≦10-18; percent identity/similarity to human IL-6: 24.74%/ 46.91%; to murine: 24.23%/ 47.94%; to porcine: 25.97%/ 52.91%; to bovine: 24.60%/ 49.73%; all alignments were calculated with the GCG software “GAP”).

[0004] The viral gene product (v-IL-6) has conserved all 4 cysteine residues that are known to be involved in IL-6 disulfide bridging, and it shows a characteristic signal peptide of 19 to 22 amino acids (FIG. 1). The area involved in binding of human IL-6 to its receptor has been mapped to the middle of the protein by two groups^(7, 8, 9). Ehlers et al. showed that amino acids 105 to 123 of the human IL-6, as shown in FIG. 1 (GFNEEtCLVKlitGLLEFE), are involved in receptor binding. Most remarkably, this region is highly conserved in v-IL-6 (GFNEtsCLkKLadGFFEFE). Identity and similarity of v-IL-6 to the receptor binding region of human IL-6 are 58% and 74%, respectively (FIG. 1). This is almost identical with the degree of conservation that can be observed in this receptor binding area of human IL-6 to murine IL-6. As both human IL-6 and murine IL-6 are able to bind to the receptor of the other species (murine IL-6 and human IL-6, respectively), it is likely that v-IL-6 is also able to bind to the human and the murine IL-6 receptor.

[0005] Rhadinoviruses frequently acquire genes from their host cell¹⁰. This HHV-8 ORF however, is the first known example of a viral IL-6 structural homologue. Up to now all cell-homologous genes of rhadinoviruses that have been tested were functional; non-functional genes would most likely have been lost in viral evolution. Thus, the conservation of essential IL-6 features makes it highly suggestive that v-IL-6 is functional in normal HHV-8 replication or persistence. Since models of paracrine growth stimulation of spindle cells by cytokines, including IL-6 and the related oncostatin M, have been proposed for KS pathogenesis, the finding of the v-IL-6 gene in HHV-8 lends support to the hypothesis that HHV-8 is causally related to this multifocal proliferation.

[0006] The present invention therefore relates to:

[0007] a) Viral interleukin-6 (v-lL-6), which can be obtained by recombinant expression of the DNA of HHV-8.

[0008] b) A polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2.

[0009] c) A fragment of v-IL-6, having the capability of binding to an IL-6 receptor and comprising the amino acid sequence GFNEtsCLkKLadGFFEFE.

[0010] d) A fragment as defined in b1, which essentially comprises the amino acid sequence GFNEtsCLkKLadGFFEFE.

[0011] e) A fragment as defined in c or d, which binds to a human IL-6 receptor.

[0012] f) A polypeptide having the amino acid sequence displayed in FIG. 2.

[0013] g) Mutants and variants of v-IL-6 or of the polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2, which mutants and variants are obtained by conventional amino acid substitutions or deletions, with the proviso that these mutants and variants are functionally equivalent to v-IL-6.

[0014] h) Fragments of v-IL-6, or of the polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2, characterized in that they are able to competively inhibit the biological activity of IL-6 in a suitable assay system.

[0015] i) An isolated nucleic acid coding for v-IL-6 or the polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2. A preferred embodiment is the nucleic acid having the nucleotide sequence of FIG. 2. Furthermore, an isolated nucleic acid, hybridizing to the abovementioned nucleic acids under stringent conditions and encoding functionally active v-IL-6 shall be comprised.

[0016] k) Monoclonal or polyclonal antibodies directed against v-lL-6 or the polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2.

[0017] l) Testkit for the detection of v-IL-6 in a sample, comprising one or more of the above monoclonal or polyclonal antibodies.

[0018] m) Testkit for the detection of antibodies against v-IL-6 comprising v-IL-6 and/or the polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2, and/or mutants and variants of v-IL-6 or the polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2 and/or fragments of v-IL-6 or the polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2.

[0019] n) Testkit for the detection of v-IL-6 DNA or RNA, comprising a nucleic acid which codes for v-IL-6, or which hybridizes to the aforementioned nucleic acid and encodes functionally active v-IL-6.

[0020] o) A medicament comprising as an active ingredient a monoclonal antibody or polyclonal antibodies directed against v-IL-6, or a polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2, or mutants, variants or fragments of v-IL-6 or the aforementioned polypeptide. In another embodiment, the medicament may comprise as an active ingredient a nucleic acid encoding v-I L-6.

[0021] p) A cell culture growth medium, comprising as an active ingredient v-IL-6 or the polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2 , or mutants, variants or fragments of v-IL-6 or the aforementioned polypeptide.

[0022] q) A process of manufacturing v-IL-6 or the polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2, or mutants and variants, or fragments of v-IL-6 or the aforementioned polypeptide.

[0023] r) A process of manufacturing a medicament, wherein the active ingredient is combined with suitable excipients and/or other auxilliary compounds according to common knowledge of those skilled in the art.

[0024] s) A process of manufacturing a medicament comprising as an active ingredient monoclonal or polyclonal antibodies directed against v-IL-6, or a polypeptide comprising v-IL-6, or mutants, variants or fragments of v-IL-6, or a nucleic acid encoding v-IL-6 for the treatment of kaposi sarcoma, Castleman's disease, multiple myeloma, kidney cell carcinoma, mesangial proliferative glomerulonephritis or B cell lymphoma.

[0025] t) A process of diagnosing an HHV-8 infection comprising the in vitro detection of v-IL-6 antigen, v-IL-6 DNA, v-IL-6 RNA or antibodies against v-IL-6.

[0026] u) A process of diagnosing the HHV-8 associated disorders kaposi sarcoma, Castleman's disease or body cavity based lymphomas (BCBL) through the diagnosis of an HHV-8 infection as described above.

[0027] v) A process of growing cells in culture, characterized in that v-IL-6 or the polypeptide, which can be obtained by recombinant expression of the DNA of HHV-8, and which comprises the amino acid sequence displayed in FIG. 2, or mutants and variants, or fragments of v-IL-6 or the aforementioned polypeptide, or mixtures of these compounds are contained in the growth medium. In a preferred process these cells are B-lymphocytes, hybridomas, hemopoetic cells or endothelial cells.

[0028] The sequence shown in FIG. 2 was generated by first subcloning shotgun fragments of lambda clone G16 into commercially available plasmid pBS KS- (Stratagene, San Diego, calif.). Resulting plasmids were purified using a commercially available kit (Qiagen, Hilden, Germany) and sequenced on an automated sequencing system (A377, Applied Biosystems GmbH, Weiterstadt, Germany) using the recommendations of the manufacturer. The sequence was determined on both strands, using standard primers for shotgun clones, and gene specific primers for further analysis. In addition to showing the coding sequence of the interleukin-6 homologue of human herpesvirus 8, the deduced amino acid sequence, in one and three letter code, is shown in the sequence listing below.

[0029] The present invention is further described in the claims.

Bibliography

[0030] 1. Miles, S. A. et al.: Science, 255,1432-1434 (1992).

[0031] 2. Stürzl, M. et al.: Oncogene 10, 2007-2016 (1995).

[0032] 3. Miles, S. A. et al.: Proc. Natl. Acad. Sci. U. S. A. 8

[0033] 4. Masood, R. et al.: AIDS Res. Hum. Retroviruses 1

[0034] 5. Chang, Y. et al.: Science. 266,1865-1869 (1994)

[0035] 6. Moore, P. S. et al.: J. Virol. 70, 549-558 (1996).

[0036] 7. Hammacher, A. et al.: Protein Sci. 3, 2280-2293

[0037] 8. Ehlers, M. et al.: J. Immunol. 153,1744-1753 (1994).

[0038] 9. Ehlers, M. et al.: Ann. N. Y. Acad. Sci. 762, 400402 (1995).

[0039] 10. Albrecht, J. C. et al.: J. Virol. 66, 5047-5058 (1992).

Legends

[0040]FIG. 1:

[0041] Alignment of the sequences of the predicted protein precursor of the HHV-8 IL-6 gene with human and mouse IL-6. Amino acids identical in all three proteins are indicated by an asterisk, cysteine residues involved in disulfide bridging are marked with an arrowhead. Upper case letters symbolize amino acids conserved according to the criteria defined by M. Dayhoff.

[0042]FIG. 2:

[0043] Nucleic acid sequence encoding v-IL-6 and corresponding amino acid sequence.

1 4 612 base pairs nucleic acid double linear DNA (genomic) CDS 1..612 1 ATG TGC TGG TTC AAG TTG TGG TCT CTC TTG CTG GTC GGT TCA CTG CTG 48 Met Cys Trp Phe Lys Leu Trp Ser Leu Leu Leu Val Gly Ser Leu Leu 1 5 10 15 GTA TCT GGA ACG CGG GGC AAG TTG CCG GAC GCC CCC GAG TTT GAA AAG 96 Val Ser Gly Thr Arg Gly Lys Leu Pro Asp Ala Pro Glu Phe Glu Lys 20 25 30 GAT CTT CTC ATT CAG AGA CTC AAT TGG ATG CTA TGG GTG ATC GAT GAA 144 Asp Leu Leu Ile Gln Arg Leu Asn Trp Met Leu Trp Val Ile Asp Glu 35 40 45 TGC TTC CGC GAC CTC TGT TAC CGT ACC GGC ATC TGC AAG GGT ATT CTA 192 Cys Phe Arg Asp Leu Cys Tyr Arg Thr Gly Ile Cys Lys Gly Ile Leu 50 55 60 GAG CCC GCT GCT ATT TTT CAT CTG AAA CTA CCA GCC ATC AAC GAT ACT 240 Glu Pro Ala Ala Ile Phe His Leu Lys Leu Pro Ala Ile Asn Asp Thr 65 70 75 80 GAT CAC TGC GGG TTA ATA GGA TTT AAT GAG ACT AGC TGC CTT AAA AAG 288 Asp His Cys Gly Leu Ile Gly Phe Asn Glu Thr Ser Cys Leu Lys Lys 85 90 95 CTC GCC GAT GGC TTT TTT GAA TTC GAG GTG TTG TTT AAG TTT TTA ACG 336 Leu Ala Asp Gly Phe Phe Glu Phe Glu Val Leu Phe Lys Phe Leu Thr 100 105 110 ACG GAG TTT GGA AAA TCA GTG ATA AAC GTG GAC GTC ATG GAG CTT CTG 384 Thr Glu Phe Gly Lys Ser Val Ile Asn Val Asp Val Met Glu Leu Leu 115 120 125 ACG AAG ACC TTA GGA TGG GAC ATA CAG GAA GAG CTC AAT AAG CTG ACT 432 Thr Lys Thr Leu Gly Trp Asp Ile Gln Glu Glu Leu Asn Lys Leu Thr 130 135 140 AAG ACG CAC TAC AGT CCA CCC AAA TTT GAC CGC GGT CTA TTA GGG AGG 480 Lys Thr His Tyr Ser Pro Pro Lys Phe Asp Arg Gly Leu Leu Gly Arg 145 150 155 160 CTT CAG GGA CTT AAG TAT TGG GTG AGA CAC TTT GCT TCG TTT TAT GTT 528 Leu Gln Gly Leu Lys Tyr Trp Val Arg His Phe Ala Ser Phe Tyr Val 165 170 175 CTG AGT GCA ATG GAA AAG TTT GCA GGT CAA GCG GTG CGT GTT TTG GAC 576 Leu Ser Ala Met Glu Lys Phe Ala Gly Gln Ala Val Arg Val Leu Asp 180 185 190 TCT ATC CCA GAC GTG ACT CCT GAC GTC CAC GAT AAG 612 Ser Ile Pro Asp Val Thr Pro Asp Val His Asp Lys 195 200 204 amino acids amino acid linear protein 2 Met Cys Trp Phe Lys Leu Trp Ser Leu Leu Leu Val Gly Ser Leu Leu 1 5 10 15 Val Ser Gly Thr Arg Gly Lys Leu Pro Asp Ala Pro Glu Phe Glu Lys 20 25 30 Asp Leu Leu Ile Gln Arg Leu Asn Trp Met Leu Trp Val Ile Asp Glu 35 40 45 Cys Phe Arg Asp Leu Cys Tyr Arg Thr Gly Ile Cys Lys Gly Ile Leu 50 55 60 Glu Pro Ala Ala Ile Phe His Leu Lys Leu Pro Ala Ile Asn Asp Thr 65 70 75 80 Asp His Cys Gly Leu Ile Gly Phe Asn Glu Thr Ser Cys Leu Lys Lys 85 90 95 Leu Ala Asp Gly Phe Phe Glu Phe Glu Val Leu Phe Lys Phe Leu Thr 100 105 110 Thr Glu Phe Gly Lys Ser Val Ile Asn Val Asp Val Met Glu Leu Leu 115 120 125 Thr Lys Thr Leu Gly Trp Asp Ile Gln Glu Glu Leu Asn Lys Leu Thr 130 135 140 Lys Thr His Tyr Ser Pro Pro Lys Phe Asp Arg Gly Leu Leu Gly Arg 145 150 155 160 Leu Gln Gly Leu Lys Tyr Trp Val Arg His Phe Ala Ser Phe Tyr Val 165 170 175 Leu Ser Ala Met Glu Lys Phe Ala Gly Gln Ala Val Arg Val Leu Asp 180 185 190 Ser Ile Pro Asp Val Thr Pro Asp Val His Asp Lys 195 200 212 amino acids amino acid <Unknown> linear protein 3 Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala Phe Ser Le 1 5 10 15 Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala Pro Val Pro Pr 20 25 30 Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu Th 35 40 45 Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly Il 50 55 60 Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys Glu Se 65 70 75 80 Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Al 85 90 95 Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys Le 100 105 110 Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Ty 115 120 125 Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala Val Gl 130 135 140 Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys As 145 150 155 160 Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser Leu Le 165 170 175 Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Thr Thr Hi 180 185 190 Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Al 195 200 205 Leu Arg Gln Met 210 211 amino acids amino acid <Unknown> linear protein 4 Met Lys Phe Leu Ser Ala Arg Asp Phe His Pro Val Ala Phe Leu Gl 1 5 10 15 Leu Met Leu Val Thr Thr Thr Ala Phe Pro Thr Ser Gln Val Arg Ar 20 25 30 Gly Asp Phe Thr Glu Asp Thr Thr Pro Asn Arg Pro Val Tyr Thr Th 35 40 45 Ser Gln Val Gly Gly Leu Ile Thr His Val Leu Trp Glu Ile Val Gl 50 55 60 Met Arg Lys Glu Leu Cys Asn Gly Asn Ser Asp Cys Met Asn Asn As 65 70 75 80 Asp Ala Leu Ala Glu Asn Asn Leu Lys Leu Pro Glu Ile Gln Arg As 85 90 95 Asp Gly Cys Tyr Gln Thr Gly Tyr Asn Gln Glu Ile Cys Leu Leu Ly 100 105 110 Ile Ser Ser Gly Leu Leu Glu Tyr His Ser Tyr Leu Glu Tyr Met Ly 115 120 125 Asn Asn Leu Lys Asp Asn Lys Lys Asp Lys Ala Arg Val Leu Gln Ar 130 135 140 Asp Thr Glu Thr Leu Ile His Ile Phe Asn Gln Glu Val Lys Asp Le 145 150 155 160 His Lys Ile Val Leu Pro Thr Pro Ile Ser Asn Ala Leu Leu Thr As 165 170 175 Lys Leu Glu Ser Gln Lys Glu Trp Leu Arg Thr Lys Thr Ile Gln Ph 180 185 190 Ile Leu Lys Ser Leu Glu Glu Phe Leu Lys Val Thr Leu Arg Ser Th 195 200 205 Arg Gln Thr 210 

1-27. (canceled)
 28. A fragment of v-IL-6 that binds an interleukin-6 (“IL-6”) receptor and consisting of the amino acid sequence (residues 87-105 of SEQ ID NO:2) GFNETSCLKKLADGFFEFE.
 29. The fragment of claim 28, which binds to a human IL-6 receptor. 